Abstract: Antibiotic resistance complicates the majority of Staphylococcus aureus (S. aureus) infections. A full two thirds of hospital-associated S. aureus infections and ~50% of those acquired in the community are now methicillin-resistant (MRSA). MRSA causes >450,000 infections in the US each year, and it is responsible for half of all deaths caused by drug-resistant bacteria. The high incidence of multi-drug resistance in S. aureus and other bacteria underscores the need for next-generation antibiotics capable of combating these dangerous pathogens. An increasingly compelling therapeutic strategy leverages recombinant enzymes, such as Staphylococcus simulans lysostaphin (LST), which degrade cell wall peptidoglycan causing bacterial lysis and death. LST is a highly potent anti-staphylococcal agent with proven efficacy against both drug-sensitive and drug-resistant strains of S. aureus. While LST holds great potential for combatting dangerous S. aureus infections, its utility as a systemically administered treatment is constrained by specific limitations. First, as a small protein of 26,942 daltons, LST is rapidly cleared from the blood stream by renal filtration. In the clinic, this fact might necessitate frequent, high dosing to achieve complete bacterial clearance. Providing the option to use lower doses and less frequent administration would reduce costs, ease patient burden, and improve treatment outcomes. Second, LST is subject to development of S. aureus resistance by virtue of mutations in the femA gene, which alters the specific peptidoglycan bond targeted by the enzyme. Synergistic 2-agent treatments have been shown to mitigate the risk of LST resistance. We propose here to construct a modular bifunctional lysin platform based on fusions with immunoglobulin Fc domains. The Fc domain is a natural bivalent display scaffold, and we aim to leverage validated knob and hole bispecific Fc engineering strategies to create heterobifunctional Fc-lysin chimeras. Specifically, we will fuse the LST catalytic and cell wall binding domains to one chain of a heterodimeric knob and hole Fc, and we will fuse the ?SA2 prophage endopeptidase domain to the second chain. The heterologous pairing of the two Fc chains will create a single molecular entity that integrates two complementary cell wall hydrolases known to exert anti-S. aureus synergy. The bifunctional Fc-lysin's two pronged attack on S. aureus cell walls should minimize acquired resistance. Additionally, the Fc domain will serve to extend the bifunctional lysin's circulation half-life by (i) increasing the molecule's size beyond the limit for first-pass glomerular filtration in the kidney, and (ii) engaging the neonatal Fc receptor (FcRn), which actively recycles IgG antibodies and promotes their exceptionally long half-lives. As a whole, this project seeks to develop a modular platform for engineering high performance antibacterial enzymes that capitalize on intramolecular synergy to kill drug-resistant bacterial pathogens.